In 2009, Ministry of the Environment, Japan, started the discussion about the implementation of bioassay to evaluate and control toxic chemical compounds in effluent and ambient water to complement the conventional chemical analysis of individual compounds, and is aimed to start the new system under Water Pollution Control Act as early as 2016. In this manuscript, the summary of the draft test manual, publicized by National Institute for Environmental Studies (NIES) in 2013, is presented, and then the current status of effluent management in other countries is summarized. There are still several difficulties in implementing the new system using bioassay such as the increase of the cost, the shortage of testing laboratories, the establishment of the benchmark, and toxicity reduction evaluation. Some of the solution measures are presented in addition to the authors' expectation for the implementation of this approach and the contribution of Reference Laboratory in NIES.

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Short-term chronic toxicity tests were conducted using a battery of three aquatic organisms (fish, daphnia, and alga) for effluents of five selected factories in Tokushima, Japan, and relatively strong toxicity was found for two factories (X and Y) for Ceriodaphnia dubia. The source water, the major processes to discharge toxicants, and their water treatment processes were examined by the interview with effluent managers. For Factory X, source water and the influent of the water treatment facility did not exert strong toxicity for C. dubia while the treated effluent was strongly toxic. As results of bio-assay for two major water additives, the inorganic additive was found to be toxic. The additional bio-assay for the major components aluminum and sulfate, and chemical analysis of these ions identified aluminum ion as a major causative agent. For Factory Y, source water was non-toxic while the influent of the water treatment facility was found to be strongly toxic. As results of interview with the effluent manager, bio-assay and chemical analysis, sulfate was found as a major causative agent. Toxicity reduction measure was under process for both factories using improvement of water treatment processes.

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The environmental effects of five industrial effluents collected from Toyama Prefecture were assessed using three short-term chronic toxicity tests using freshwater organisms. All effluents were found to be toxic. Among the three tests, the crustacean and algal tests with maximum chronic toxicity unit (TUc) values more than 320 and 10, respectively, were more sensitive than the fish test. In two samples, toxicity characterization tests indicated that the adverse effects were due to the presence of heavy metals and ammonia. Furthermore, a field and on-site survey was conducted for one discharger, the effluent from which had been found to contain toxic levels of Ni. Two in-stream samples collected downstream from the discharge point showed chronic toxicity in algal and crustacean tests, although the samples collected upstream from the discharge point did not show any adverse effects. The heavy metal removal efficiency of on-site treatment facilities was assessed to examine the possibility of reducing the effluent toxicity. Among the four on-site treatment facilities that processed the effluents comprising the total discharge, one showed relatively lower Ni removal efficiency compared with the other facilities. These results suggest that improvement in the heavy metal removal efficiency of this facility is crucial for reducing the toxic effect of the effluents.

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Compliance with chemical-based effluent standards does not always ensure ecological risk management of industrial effluent which contains a variety of chemicals used in a chemical manufacturer. To evaluate the biological effects of effluent discharged from our factories, we have applied Whole Effluent Toxicity (WET) test on effluent management system since 2007. Effluent samples were collected at least once a year from four factories and subjected to a battery of bioassays using fish, daphnids, algae, and luminescence bacteria. Our results showed that the most sensitive test species demonstrating toxic effects including annual changes were different for the four effluents. Considering the dilution factor of effluent after discharge, the ecological risk of all samples, which includes the highest toxic effluent from factory D, was assumed negligible in the receiving waters. To reduce effluent toxicity in factory D, we investigated the weekly and daily fluctuation of the effluent toxicity, suggesting that the effluent toxicity was different depending on products. Toxicity assessment of effluent samples collected from each drainage system inside the factory D identified the in-plant source of toxicity, which could be suppressed by reducing the amount of sodium hypochlorite. Moreover, C18 resin treatment in lab-scale reduced the toxicity of the effluent to the daphnids, indicating that at least some of the toxic chemicals were organic compounds.

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Whole Effluent Toxicity (WET) testing evaluates the toxic effect of unknown or multiple substances in wastewater using aquatic organisms. It has already been implement in the US. When the result of WET testing indicates the effect exceeding a certain standard, Toxicity Reduction Evaluation (TRE) and Toxicity Identification Evaluation (TIE) are required for identifying the effluent characteristics causing toxicity and causing the toxicity.
In this study, we evaluated effluents from the metal manufacturing plant using three types of aquatic organisms (Danio rerio, Ceriodaphnia dubia, and Pseudokirchneriella subcapitata) with reference to the draft testing methods for effluent using bioassays. The result for one of effluents indicated toxic effect on C. dubia.
We conducted chelate resin absorption test and flocculation test as TRE/TIE. As a result, we identified Zn and Ni in the effluent as possible major toxicants and studied improvement of wastewater treatment for reducing toxicity.

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In view of developing a bioassay for use into effluent management system in Japan, short-term toxicity tests using fish, daphnids, and algae were selected as a battery of bioassays, which evaluate the integrated effect of effluent, and "a draft protocol of bioassays for effluent testing" has been prepared. To examine the robustness, sensitivity and reproducibility of the draft protocol, we conducted an inter-laboratory trial where the same effluent was tested by 9 laboratories in Japan. No observed effect concentrations (NOEC) of the test effluent in all the laboratories were within the median +/-1 dilutions, suggesting that the variability between laboratories was at acceptable level. Furthermore, testing of reference chemicals by 10 testing laboratories showed that the reproducibility of the bioassays was relatively higher than that exhibited in the inter-laboratory validation of similar bioassays conducted by US EPA. However, the percentage of minimum significant difference (PMSD) and control coefficient of variation (CV) in the almost all the Japanese laboratories were at acceptable levels (as appears in the US EPA report), indicating that the intra-laboratory precision and the sensitivity of each test was sufficient. We investigated the value of using IC₂₀ for fish, IC₂₅ for daphnids, and IC₅ for algae as an alternative to NOEC. With this approach, toxicity survey of 91 effluent samples conducted from 2008 to 2013 found that 36% of effluents were toxic to at least one of the three test species even after 10-fold dilution. The test species that demonstrated toxic effects were different in each sample; thus, we suggest that effluent toxicity evaluation should be based on more than one species due to different sensitivities.

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The current worldwide regulatory guideline requires evaluation of the chronic effects of chemicals on water flea Daphnia magna. Based on the life cycle of D.magna, chronic tests with survival and reproductive endpoints requires a 21 days exposure period. Ceriodaphnia dubia, a common freshwater invertebrate similar to the D.magna, has a shorter life cycle and therefore reproductive (and survival) endpoints have been established following about 8 days of exposure. No observed effect concentrations for reproduction were obtained for D.magna and C.dubia following exposure to general 10 organic compounds (ie. aromatic amines as pharmaceutical, chlorinated aromatic compound, polyaromatic compound and alkyl ester) according to the OECD TG 211 and the biological test method of Environmental Canada. These data were evaluated to determine whether one organism might be considered more sensitive. Reproduction data obtained from C.dubia chronic test provide similar sensitivity to D.magna in this study. Based on these data, C.dubia may be used as a cost-effective alternative and representative invertebrate species when assessing the potential risk on the organic compounds.

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